This invention relates to a process for the preparation of an enzymatically active product.
Enzymatic reactions are normally carried out with one or more enzymes in solution in the medium containing the substance which it is desired to subject to the action of the enzyme and which is known as the substrate. Thus dissolved in the reaction medium, the enzyme can only be separated from the substrate with extreme difficulty and generally the enzyme has to be inactivated, for example by heating, in order to stop the reaction. Accordingly, this enzyme is not reusable.
Numerous processes for the preparation of enzymatically active products insoluble in aqueous medium have been proposed with a view to obviating these disadvantages. The enzymatic reactions carried out with these products may be conducted either by percolation of the substrate through a column filled with insoluble product or by dispersion of the product in the solution containing the substrate and by mechanical separation of the insoluble product. In every case, the insoluble, enzymatically active product is readily recoverable and, at least in theory, indefinitely reusable.
However, there are serious limitations to the use of these insoluble enzymatically active products, because the successful execution of a chemical, i.e. enzymatic, reaction requires that a certain number of conditions be satisfied.
The first requirement, which is imperative, is that the reactants should be able readily to contact one another, i.e. in the present case that the substrate should have fairly free access to the insoluble enzymatically active product and, more especially, to the active site. Accordingly, it is essential that there should be, at one and the same time, a high level of physico-chemical affinity between the two reactants present, reduced steric congestion around the active site and, concurrently or correlatively, a certain flexibility of the enzymatically active product. Although this first requirement of "good contact" might be satisfied, the enzyme-substrate exchange rate also has to be suitable and must ensure the rapid departure of one molecule of substrate which has reacted and its immediate replacement by a molecule which has not yet reacted.
The conditions under which the reaction between the substrate and the insoluble enzymatically active product is carried out are generally unfavourable. Accessibility and exchange rate are poor as a result of the heterogeneity of the reaction medium (solid-liquid), the poor accessibility being aggravated by the fact that the product is a solid polymer and, hence, shows a pronounced degree of rigidity. In addition, this polymer frequently comprises a hydrocarbon-containing skeleton which shows little affinity on the one hand for the aqueous phase (poor wettability) and on the other hand for the substrates which are generally polar.
Accordingly, the insoluble products normally have a low enzymatic activity by comparison with the corresponding free enzyme and, hence give mediocre reaction yields. An object of the invention is to eliminate these limitations.